This invention relates to biological reagents which comprise compounds that inhibit xcex2-amyloid peptide release and/or its synthesis, and, accordingly, have utility in determining the cellular mechanism involved in the generation of xcex2-amyloid peptide.
The following publications, patents and patent applications are cited in this application as superscript numbers:
1 Glenner, et al., xe2x80x9cAlzheimer""s Disease: Initial Report of the Purification and Characterization of a Novel Cerebrovascular Amyloid Proteinxe2x80x9d, Biochem. Biophys. Res. Commun., 120:885-890 (1984).
2 Glenner, et al., xe2x80x9cPolypeptide Marker for Alzheimer""s Disease and its Use for Diagnosisxe2x80x9d, U.S. Pat. No. 4,666,829 issued May 19, 1987.
3 Selkoe, xe2x80x9cThe Molecular Pathology of Alzheimer""s Diseasexe2x80x9d, Neuron, 6:487-498 (1991).
4 Goate, et al., xe2x80x9cSegregation of a Missense Mutation in the Amyloid Precursor Protein Gene with Familial Alzheimer""s Diseasexe2x80x9d, Nature, 349:704-706 (1990).
5 Chartier-Harlan, et al., xe2x80x9cEarly-Onset Alzheimer""s Disease Caused by Mutations at Codon 717 of the xcex2-Amyloid Precursor Proteing Genexe2x80x9d, Nature, 353:844-846 (1989).
6 Murrell, et al., xe2x80x9cA Mutation in the Amyloid Precursor Protein Associated with Hereditary Alzheimer""s Diseasexe2x80x9d, Science, 254:97-99 (1991).
7 Mullan, et al., xe2x80x9cA Pathogenic Mutation for Probable Alzheimer""s Disease in the APP Gene at the N-Terminus of xcex2-Amyloid, Nature Genet., 1:345-347 (1992).
8 International Patent Application No. PCT/US97/20356; Publication No. WO98/22441
9 International Patent Application No. PCT/US97/20355; Publication No. WO98/22430
0 International Patent Application No. PCT/US97/18704; Publication No. WO98/22493
111 International Patent Application No. PCT/US97/20804; Publication No. WO98/22494
12 Bioconjugate Chemistry (1990) 1(6) 431-437
13 International Patent Application No. PCT/US97/22986; Publication No. WO98/28268
All of the above publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
Alzheimer""s Disease (AD) is a degenerative brain disorder characterized clinically by progressive loss of memory, cognition, reasoning, judgment and emotional stability that gradually leads to profound mental deterioration and ultimately death. AD is a very common cause of progressive mental failure (dementia) in aged humans and is believed to represent the fourth most common medical cause of death in the United States. AD has been observed in races and ethnic groups worldwide and presents a major present and future public health problem. The disease is currently estimated to affect about two to three million individuals in the United States alone. AD is at present incurable. No treatment that effectively prevents AD or reverses its symptoms and course is currently known.
The brains of individuals with AD exhibit characteristic lesions termed senile (or amyloid) plaques, amyloid angiopathy (amyloid deposits in blood vessels) and neurofibrillary tangles. Large numbers of these lesions, particularly amyloid plaques and neurofibrillary tangles, are generally found in several areas of the human brain important for memory and cognitive function in patients with AD. Smaller numbers of these lesions in a more restrictive anatomical distribution are also found in the brains of most aged humans who do not have clinical AD. Amyloid plaques and amyloid angiopathy also characterize the brains of individuals with Trisomy 21 (Down""s Syndrome) and Hereditary Cerebral Hemorrhage with Amyloidosis of the Dutch Type (HCHWA-D). At present, a definitive diagnosis of AD usually requires observing the aforementioned lesions in the brain tissue of patients who have died with the disease or, rarely, in small biopsied samples of brain tissue taken during an invasive neurosurgical procedure.
The principal chemical constituent of the amyloid plaques and vascular amyloid deposits (amyloid angiopathy) characteristic of AD and the other disorders mentioned above is an approximately 4.2 kilodalton (kD) protein of about 39-43 amino acids designated the xcex2-amyloid peptide (xcex2AP) or sometimes Axcex2, Axcex2P or xcex2/A4. xcex2-Amyloid peptide was first purified and a partial amino acid sequence was provided by Glenner, et al.1 The isolation procedure and the sequence data for the first 28 amino acids are described in U.S. Pat. No. 4,666,8292.
Molecular biological and protein chemical analyses have shown that the xcex2-amyloid peptide is a small fragment of a much larger precursor protein (APP), that is normally produced by cells in many tissues of various animals, including humans. Knowledge of the structure of the gene encoding the APP has demonstrated that xcex2-amyloid peptide arises as a peptide fragment that is cleaved from APP by protease enzymes. The precise biochemical mechanism by which the xcex2-amyloid peptide fragment is cleaved from APP and subsequently deposited as amyloid plaques in the cerebral tissue and in the walls of the cerebral and meningeal blood vessels is currently unknown.
Several lines of evidence indicate that progressive cerebral deposition of xcex2-amyloid peptide plays a seminal role in the pathogenesis of AD and can precede cognitive symptoms by years or decades. See, for example, Selkoe3. The most important line of evidence is the discovery that missense DNA mutations at amino acid 717 of the 770-amino acid isoform of APP can be found in affected members but not unaffected members of several families with a genetically determined (familial) form of AD (Goate, et al.4; Chartier Harlan, et al.5; and Murrell, et al.6) and is referred to as the Swedish variant. A double mutation changing lysine595-methionine596 to asparagine595-leucine596 (with reference to the 695 isoform) found in a Swedish family was reported in 1992 (Mullan, et al.7). Genetic linkage analyses have demonstrated that these mutations, as well as certain other mutations in the APP gene, are the specific molecular cause of AD in the affected members of such families. In addition, a mutation at amino acid 693 of the 770-amino acid isoform of APP has been identified as the cause of the xcex2-amyloid peptide deposition disease, HCHWA-D, and a change from alanine to glycine at amino acid 692 appears to cause a phenotype that resembles AD is some patients but HCHWA-D in others. The discovery of these and other mutations in APP in genetically based cases of AD prove that alteration of APP and subsequent deposition of its xcex2-amyloid peptide fragment can cause AD.
Despite the progress which has been made in understanding the underlying mechanisms of AD and other xcex2-amyloid peptide related diseases, there still remains a need to determine the precise mechanism for the generation of xcex2-amyloid peptide. Biological reagents comprising compounds known to inhibit the generation of xcex2-amyloid peptide would be useful in determining the mechanism for the generation of xcex2-amyloid peptide and thus AD. Knowledge of the disease mechanism would, in turn, allow rationale drug design of novel entities which specifically target AD.
This invention is directed to the discovery of a class of biological reagents which comprise compounds that inhibit xcex2-amyloid peptide release and/or its synthesis and, therefore, are useful in determining the underlying cellular mechanism leading to AD in patients susceptible to AD. The class of biological reagents having the described properties are defined by formula I below:
Axe2x80x94Bxe2x80x94Cxe2x80x83xe2x80x83I
wherein:
A is selected from the group consisting of formulas II, III, IV and V below;
B is selected from the group consisting of
a) (CH2CH(R)Q)n 
b) alkylene-Q
c) substituted alkylene-Q
wherein R is selected from hydrogen, alkyl, aryl and Q is selected from the group consisting of xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94OC(O)NHxe2x80x94, xe2x80x94NHC(O)Oxe2x80x94, xe2x80x94NHC(O)NHxe2x80x94, xe2x80x94NHC(O)xe2x80x94 and xe2x80x94C(O)NHxe2x80x94; and
C is selected from the group consisting of a solid support and a detectable marker wherein C is optionally linked to Q through a linking arm;
wherein Formula II is defined as follows: 
wherein R11 is selected from the group consisting of:
(a) a substituted phenyl group of the formula: 
xe2x80x83wherein
Rc is selected from the group consisting of acyl, alkyl, alkoxy, alkoxycarbonyl, alkylalkoxy, azido, cyano, halo, hydrogen, nitro, trihalomethyl, thioalkoxy, and wherein Rb and Rc are fused to form a heteroaryl or heterocyclic ring with the phenyl ring wherein the heteroaryl or heterocyclic ring contains from 3 to 8 atoms of which from 1 to 3 are heteroatoms independently selected from the group consisting of oxygen, nitrogen and sulfur;
Rb and Rbxe2x80x2 are independently selected from the group consisting of hydrogen, halo, nitro, cyano, trihalomethyl, alkoxy, and thioalkoxy with the proviso that Rb, Rbxe2x80x2 and Rc are not all hydrogen and with the further proviso that when RC is hydrogen, then neither Rb nor Rbxe2x80x2 are hydrogen;
(b) 2-naphthyl; and
(c) 2-naphthyl substituted at the 4, 5, 6, 7 and/or 8 positions with 1 to 5 substituents selected from the group consisting of alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy;
R12 is selected from the group consisting of hydrogen, alkyl of from 1 to 4 carbon atoms, alkylalkoxy of from 1 to 4 carbon atoms and alkylthioalkoxy of from 1 to 4 carbon atoms; and
R13 is selected from the group consisting of xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94Oxe2x80x94(CH2)pC(O)xe2x80x94, xe2x80x94Oxe2x80x94(CH2)pC(O)Oxe2x80x94 and xe2x80x94Oxe2x80x94(CH2)pC(O)NHxe2x80x94, wherein p is an integer of from 1 to 2;
wherein Formula III is defined as follows: 
wherein R21 is selected from the group consisting of
a) alkyl, alkenyl, alkcycloalkyl, phenyl-(Rd)m-, naphthyl-(Rd)m- wherein Rd is an alkylene group of from 1 to 8 carbon atoms and m is an integer equal to 0 or 1, cycloalkyl, cycloalkenyl, 3-pyridyl, 4-pyridyl and heteroaryl, other than 3- and 4-pyridyl, of 3 to 10 atoms and 1 to 4 heteroatoms selected from oxygen, sulfur and nitrogen wherein the heteroaryl group is optionally substituted with 1 to 3 substituents selected from the group consisting of alkyl, alkoxy, aryl, aryloxy, halo, nitro, thioalkoxy, and thioaryloxy with the proviso that for such heteroaryls when there is at least one nitrogen heteroatom, there is also at least one oxygen and/or sulfur heteroatom;
(b) a substituted phenyl group of the formula: 
xe2x80x83wherein
R is alkylene of from 1 to 8 carbon atoms,
m is an integer equal to 0 or 1,
Re and Rf are independently selected from the group consisting of hydrogen, hydroxy, fluoro and methyl;
Rg and Rh are independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, cyano, cycloalkyl, halo, heteroaryl, heterocyclic, nitro, trihalomethyl, thioalkoxy, thioaryloxy, thioheteroaryloxy, and xe2x80x94C(O)Rj where Rj is selected from the group consisting of alkyl, aryl, alkoxy and aryloxy; and
Ri is selected from the group consisting of hydrogen, alkyl, aryl, cyano, halo, nitro, and where Rg and Ri are fused to form a methylenedioxy ring with the phenyl ring; and
when Rg and/or Rh and/or Ri is fluoro, chloro, bromo and/or nitro, then Re and/or Rf can also be chloro; and
(c) 1- or 2-naphthyl-(Rk)m-substituted at the 5, 6, 7 and/or 8 positions with 1 to 4 substituents selected from the group consisting alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy wherein Rk is an alkylene group of from 1 to 8 carbon atoms and m is an integer equal to 0 or 1;
R22 is selected from the group consisting of hydrogen, alkyl, phenyl, alkylalkoxy, alkylthioalkoxy;
X is oxygen or sulfur;
Xxe2x80x2 is hydrogen, hydroxy or fluoro; and
Xxe2x80x3 is hydrogen, hydroxy or fluoro, or Xxe2x80x2 and Xxe2x80x3 together form an oxo group;
wherein Formula IV is defined as follows: 
wherein:
R31 is selected from the group consisting of
(a) phenyl,
(b) a substituted phenyl group of the formula: 
xe2x80x83wherein
Rp is selected from the group consisting of acyl, alkyl, alkoxy, alkylalkoxy, azido, cyano, halo, hydrogen, nitro, trihalomethyl, thioalkoxy, and wherein Rp and Rm are fused to form a heteroaryl or heterocyclic ring with the phenyl ring,
Rm and Rn are independently selected from the group consisting of hydrogen, halo, nitro, cyano, trihalomethyl, alkoxy, and thioalkoxy with the proviso that when Rp is hydrogen, then Rm and Rn are either both hydrogen or both substituents other than hydrogen,
(c) 2-naphthyl,
(d) 2-naphthyl substituted at the 4, 5, 6, 7 and/or 8 positions with 1 to 5 substituents selected from the group consisting of alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, thioalkoxy, aryl, and heteroaryl,
(e) heteroaryl, and
(f) substituted heteroaryl containing 1 to 3 substituents selected from the group consisting of alkyl, alkoxy, aryl, aryloxy, cyano, halo, nitro, heteroaryl, thioalkoxy and thioaryloxy provided that said substituents are not ortho (adjacent) to the heteroaryl attachment to the xe2x80x94NH group;
R32 is selected from the group consisting of hydrogen, alkyl of from 1 to 4 carbon atoms, alkylalkoxy of from 1 to 4 carbon atoms, alkylthioalkoxy of from 1 to 4 carbon atoms, aryl, heteroaryl, substituted aryl and substituted heteroaryl provided that the substituents are not ortho (adjacent) to the attachment of the aryl or heteroaryl atom to the carbon atom;
R33 is selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, substituted alkyl, substituted alkenyl, substituted alkynyl, and heterocyclic; and
X is selected from the group consisting of oxygen and sulfur; and
wherein Formula V is defined as follows: 
wherein
R41 is selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkenyl, substituted alkynyl, aryl, heteroaryl and heterocyclic;
R42 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclic;
each R43 is independently selected from the group consisting of hydrogen and methyl and R43 together with R44 can be fused to form a cyclic structure of from 3 to 8 atoms which is optionally fused with an aryl or heteroaryl group;
each R44 is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, heterocyclic, substituted alkyl, substituted alkenyl and substituted alkynyl;
each R45 is selected from hydrogen and methyl or together with R43 forms a cycloalkyl group of from 3 to 6 carbon atoms;
X is selected from oxygen, sulfur and NH;
Xxe2x80x2 is hydrogen, hydroxy or fluoro; and
Xxe2x80x3 is hydrogen, hydroxy or fluoro, or Xxe2x80x2 and Xxe2x80x3 together form an oxo group;
Z is selected from the group consisting of a bond covalently linking R41 to xe2x80x94CXxe2x80x2Xxe2x80x3xe2x80x94, oxygen and sulfur; and
n is an integer equal to 1 to 3;
wherein the compounds of formulas II, III, IV and V are effective in inhibiting the cellular release and/or synthesis of xcex2-amyloid peptide.
Accordingly, in one of its method aspects, this invention is directed to a method for determining the proteins involved in xcex2-amyloid peptide release and/or its synthesis in a cell which method comprises contacting the components of a cell with an effective amount of a biological reagent or a mixture of biological reagents of formula I above.
In another of its composition aspects, this invention is directed to intermediates useful in the synthesis of compounds of formula I wherein said intermediates are represented by formula VI
Axe2x80x94Bxe2x80x2xe2x80x83xe2x80x83VI
wherein:
A is selected from the group consisting of formulas II, III, IV and V as described above; and
Bxe2x80x2 is selected from the group consisting of
a) (CH2CH(R)Q)nH
b) alkylene-Q-H
c) substituted alkylene-Q-H
wherein R is selected from hydrogen, alkyl, aryl and Q is selected from the group consisting of xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94NHxe2x80x94.
Suitable compounds described by formula I and VI above include, by way of example, the following:
Tert-butyl N-(8-amino-3,6-dioxaoctyl) carbamate
Methyl N-2-aminoethyl-Nxe2x80x2-2-t-butylcarbamoylethyl amine
Tert-butyl N-(8-Nxe2x80x2-(Nxe2x80x3-benzyloxycarbonyl-L-phenylglycine)-3,6-dioxaoctyl) carbamate
Methyl N-(2-Nxe2x80x2-(Nxe2x80x3-benzyloxycarbamoyl-L-phenylglycine)ethyl)-Nxe2x80x2-2-t-butylcarbamoylethyl amine
Tert-butyl N-(2-Nxe2x80x2-(Nxe2x80x3-benzyloxycarbonyl-L-phenylglycine)-ethyl) carbamate
Tert-butyl N-(8-Nxe2x80x2L-phenylglycine-3,6-dioxaoctyl) carbamate
Methyl N-(2-N-(L-phenylglycine)ethyl)-Nxe2x80x2-2-t-butylcarbamoylethyl amine
Tert-butyl N-(2-Nxe2x80x2-L-phenylglycine)-ethyl carbamate
Tert-butyl N-[8-Nxe2x80x2-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-alaninyl)-L-phenylglycine)-3,6 -dioxaoctyl] carbamate
Methyl N-[2-N-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-alaninyl)-L-phenylglycine)ethyl]-Nxe2x80x2-2-t-butylcarbamoylethyl amine
Tert-butyl N-[2-Nxe2x80x2-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-alaninyl)-L-phenylglycine)-ethyl]carbamate
Tert-butyl-2-(2xe2x80x2-(Nxe2x80x2-benzyloxycarbonyl-L-phenylglycine) aminoethoxy)ethylcarbamate
Tert-butyl-2-(2xe2x80x2-(L-phenylglycine)aminoethoxy)ethylcarbamate.
N-((R/S)-3,5-difluorophenyl-xcex1-hydroxyacetyl)-L-alanine, methyl ester
N-((R/S -3,5-difluorophenyl-xcex1-hydroxyacetyl)-L-alanine
Tert-butyl-2-(2xe2x80x2-(Nxe2x80x2-(((R/S)-3,5-difluorophenyl-xcex1-hydroxyacetyl)-L-alanine)-L-phenylglycine)aminoethoxy)ethylcarbamate
8-Nxe2x80x2-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-alaninyl)-L-phenylglycinyl)amino-3,6-dioxaoctylamine hydrochloride
Methyl N-[2-N-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-alaninyl)-L-phenylglycinyl)aminoethyl]-Nxe2x80x2-2-aminoethyl amine hydrochloride
2-Nxe2x80x2-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-alaninyl)-L-phenylglycine)-ethylamine hydrochloride
N-(8-Nxe2x80x2-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-alaninyl)-L-phenylglycine)-3,6-dioxaoctyl)-biotinamide
2-(2xe2x80x2-(Nxe2x80x2-(((R/S)-3,5-difluorophenyl-xcex1-hydroxyacetyl)-L-alanine)-L-phenylglycine)aminoethoxy)ethylamine, hydrochloride salt
1-[N-(3,5-difluorophenylacetyl)-L-alaninyl)-(L-phenylglycinyl)]amino-6-(N-biotinyl)aminohexane
1-[N-(3 ,5-difluorophenylacetyl)-L-alaninyl)-(L-phenylglycinyl)]amino-3-(4-iodophenoxy)propane
1-[N-(3 ,5-difluorophenylacetyl)-L-alaninyl)-(L-phenylglycinyl)]amino-6-[N-(4-amino-7-nitrobenzofurazanyl)]hexane
1-[N-(3,5-difluorophenylacetyl)-L-alaninyl)-(L-phenylglycinyl)]amino-3-(4-trimethylstannylphenoxy) propane
N-{1-N-[N-(3,5-difluorophenylacetyl)-L-alaninyl-(L-phenylglycinyl)amino-6-hexyl]}-4,4-difluoro-5,7-dimethyl-4-bora-3xcex1,4xcex1-diaza-s-indacene-3-propionamide
N-{methyl N-[2-N-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-alaninyl)-L-phenylglycinyl)aminoethyl]-Nxe2x80x2-2-aminoethyl}-4,4-difluoro-5,7-dimethyl-4-bora-3xcex1,4xcex1-diaza-s-indacene-3-propionamide
N-[8-Nxe2x80x2-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-alaninyl)-L-phenylglycinyl)amino-3,6-dioxaoctyl]-4,4-difluoro-5,7-dimethyl-4-bora-, 3xcex1,4xcex1-diaza-s-indacene-3-propionamide
5-(S)-[Nxe2x80x2-((S)-3,5-Difluorophenyl-xcex1-hydroxyacetyl)-L-alaninyl]amino-7-methyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one
5-(S)-[Nxe2x80x2-((R)-3,5-Difluorophenyl-xcex1-hydroxyacetyl)-L-alaninyl]amino-7-methyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one
5-(S)-(Nxe2x80x2-((S)-(+)-2-Hydroxy-3-methylbutyryl)-L-alaninyl)amino-7-methyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one
1-[N-(3,5-difluorophenylacetyl)-L-alaninyl)-(L-phenylglycinyl)]amino-4-p-azidosalicylamidobutane
As above, this invention relates to biological reagents comprising compounds which inhibit xcex2-amyloid peptide release and/or its synthesis, and, accordingly, have utility in determining the mechanism for the generation of xcex2-amyloid peptide. However, prior to describing this invention in further detail, the following terms will first be defined.
Definitions
The term xe2x80x9cxcex2-amyloid peptidexe2x80x9d refers to a 39-43 amino acid peptide having a molecular weight of about 4.2 kD, which peptide is substantially homologous to the form of the protein described by Glenner, et al.1 including mutations and post-translational modifications of the normal xcex2-amyloid peptide. In whatever form, the xcex2-amyloid peptide is approximately a 39-43 amino acid fragment of a large membrane-spanning glycoprotein, referred to as the xcex2-amyloid precursor protein (APP). Its 43-amino acid sequence is:
1
Asp Ala Glu Phe Arg His Asp Ser Gly Tyr
11
Glu Val His His Gln Lys Leu Val Phe Phe
21
Ala Glu Asp Val Gly Ser Asn Lys Gly Ala
31
Ile Ile Gly Leu Met Val Gly Gly Val Val
41
Ile Ala Thr (SEQ ID NO: 1)
or a sequence which is substantially homologous thereto.
xe2x80x9cAlkylxe2x80x9d refers to monovalent alkyl groups preferably having from 1 to 10 carbon atoms and more preferably 1 to 6 carbon atoms. This term is exemplified by groups such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, n-hexyl, and the like.
xe2x80x9cAlkylenexe2x80x9d refers to divalent alkylene groups preferably having from 1 to 10 carbon atoms and more preferably 1to 6 carbon atoms. This term is exemplified by groups such as methylene (xe2x80x94CH2xe2x80x94), ethylene (xe2x80x94CH2CH2xe2x80x94), the propylene isomers (e.g., xe2x80x94CH2CH2CH2xe2x80x94 and xe2x80x94CH(CH3)CH2xe2x80x94) and the like.
xe2x80x9cAlkarylxe2x80x9d refers to -alkylene-aryl groups preferably having from 1 to 10 carbon atoms in the alkylene moiety and from 6 to 10 carbon atoms in the aryl moiety. Such alkaryl groups are exemplified by benzyl, phenethyl and the like.
xe2x80x9cAlkoxyxe2x80x9d refers to the group xe2x80x9calkyl-Oxe2x80x94xe2x80x9d where alkyl is as defined herein. Preferred alkoxy groups include, by way of example, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.
xe2x80x9cAlkoxycarbonylxe2x80x9d refers to the group xe2x80x9calkyl-Oxe2x80x94C(O)xe2x80x94xe2x80x9d wherein alkyl is as defined herein. Such groups include, by way of example, methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, iso-propoxycarbonyl, n-butoxycarbonyl, tert-butoxycarbonyl, sec-butoxycarbonyl, n-pentoxycarbonyl, n-hexoxycarbonyl, and the like.
xe2x80x9cAlkylalkoxyxe2x80x9d refers to the group xe2x80x9c-alkylene-O-alkylxe2x80x9d wherein alkylene and alkoxy are as defined herein. Such groups include, by way of example, methylmethoxy (xe2x80x94CH2OCH3), ethylmethoxy (xe2x80x94CH2CH2OCH3), n-propyl-iso-propoxy (xe2x80x94CH2CH2CH2OCH(CH3)2), methyl-tert-butoxy (xe2x80x94CH2xe2x80x94Oxe2x80x94C(CH3)3) and the like.
xe2x80x9cAlkylthioalkoxyxe2x80x9d refers to the group xe2x80x9c-alkylene-S-alkylxe2x80x9d wherein alkylene and alkoxy are as defined herein. Such groups include, by way of example, methylthiomethoxy (xe2x80x94CH2SCH3), ethylthiomethoxy (xe2x80x94CH2CH2SCH3), n-propyl-iso-thiopropoxy (xe2x80x94CH2CH2CH2SCH(CH3)2), methyl-tert-thiobutoxy (xe2x80x94CH2SC(CH3)3) and the like.
xe2x80x9cAlkenylxe2x80x9d refers to alkenyl groups preferably having from 2 to 10 carbon atoms and more preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-2 sites of alkenyl unsaturation. Preferred alkenyl groups include ethenyl (xe2x80x94CHxe2x95x90CH2), n-propenyl (xe2x80x94CH2CHxe2x95x90CH2), iso-propenyl (xe2x80x94C(CH3)xe2x95x90CH2), but-2-enyl (xe2x80x94CH2CHxe2x95x90CHCH3) and the like.
xe2x80x9cAlkynylxe2x80x9d refers to alkynyl groups preferably having from 2 to 10 carbon atoms and more preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-2 sites of alkynyl unsaturation. Preferred alkynyl groups include ethynyl (xe2x80x94Cxe2x89xa1CH), propargyl (xe2x80x94CH2Cxe2x89xa1CH) and the like.
xe2x80x9cAcylxe2x80x9d refers to the groups alkyl-C(O)xe2x80x94, aryl-C(O)xe2x80x94, and heteroaryl-C(O)xe2x80x94where alkyl, aryl and heteroaryl are as defined herein.
xe2x80x9cAcylaminoxe2x80x9d refers to the group xe2x80x94C(O)NRR where each R is independently hydrogen or alkyl where alkyl is as defined herein.
Alkcycloalkylxe2x80x9d refers to the group -alkylene-cycloalkyl wherein alkylene and cycloalkyl are as defined herein.
xe2x80x9cAminoacylxe2x80x9d refers to the group xe2x80x94NRc(O)R where each R is independently hydrogen or alkyl where alkyl is as defined herein.
xe2x80x9cAcyloxyxe2x80x9d refers to the groups alkyl-C(O)Oxe2x80x94, aryl-C(O)Oxe2x80x94, heteroaryl-C(O)Oxe2x80x94, and heterocyclic-C(O)Oxe2x80x94 where alkyl, aryl, heteroaryl and heterocyclic are as defined herein.
xe2x80x9cAminoacyloxyxe2x80x9d refers to the groups xe2x80x94NRC(O)O-alkyl, xe2x80x94NRC(O)O-substituted alkyl, xe2x80x94NRC(O)O-cycloalkyl, xe2x80x94NRC(O)O-aryl, xe2x80x94NRC(O)O-heteroaryl-, and xe2x80x94NRC(O)O-heterocyclic where R is hydrogen, alkyl, substituted alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclic and where each of alkyl, substituted alkyl, cycloalkyl, aryl, heteroaryl and heterocyclic are as defined herein.
xe2x80x9cOxyacylaminoxe2x80x9d refers to the groups xe2x80x94OC(O)NR-alkyl, xe2x80x94OC(O)NR-substituted alkyl, xe2x80x94OC(O)NR-aryl, xe2x80x94OC(O)NR-heteroaryl-, and xe2x80x94OC(O)NR-heterocyclic where R is hydrogen, alkyl, substituted alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclic and where each of alkyl, substituted alkyl, cycloalkyl, aryl, heteroaryl and heterocyclic are as defined herein.
xe2x80x9cArylxe2x80x9d refers to an unsaturated aromatic carbocyclic group of from 6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl). Preferred aryls include phenyl, naphthyl and the like.
Unless otherwise constrained by the definition for the aryl substituent, such aryl groups can optionally be substituted with from 1 to 3 substituents selected from the group consisting of hydroxy, acyl, acyloxy, alkyl, alkoxy, alkenyl, alkynyl, amino, aminoacyl, aryl, aryloxy, carboxyl, alkoxycarbonyl, acylamino, cyano, halo, nitro, heteroaryl, trihalomethyl and the like. Preferred substituents include alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy.
xe2x80x9cAryloxyxe2x80x9d refers to the group aryl-Oxe2x80x94 wherein the aryl group is as defined above including optionally substituted aryl groups as also defined above.
xe2x80x9cCaboxylalkylxe2x80x9d refers to the group xe2x80x94C(O)(O)-alkyl where alkyl is as defined herein.
xe2x80x9cCycloalkylxe2x80x9d refers to cyclic alkyl groups of from 3 to 10 carbon atoms having a single cyclic ring or multiple condensed rings which can be optionally substituted with from 1 to 3 alkyl groups. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, 1-methylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.
xe2x80x9cCycloalkenylxe2x80x9d refers to cyclic alkenyl groups of from 4 to 8 carbon atoms having a single cyclic ring and at least one point of internal unsaturation which can be optionally substituted with from 1 to 3 alkyl groups. Examples of suitable cycloalkenyl groups include, for instance, cyclobut-2-enyl, cyclopent-3-enyl, cyclooct-3-enyl and the like.
xe2x80x9cHaloxe2x80x9d or xe2x80x9chalogenxe2x80x9d refers to fluoro, chloro, bromo and iodo and preferably is either chloro or fluoro.
xe2x80x9cHeteroarylxe2x80x9d refers to a monovalent aromatic group of from 2 to 8 carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen and sulfur within the ring.
Unless otherwise constrained by the definition for the heteroaryl substituent, such heteroaryl groups can be optionally substituted with 1 to 3 substituents selected from the group consisting of alkyl, alkoxy, aryl, aryloxy, halo, nitro, heteroaryl, thioalkoxy, thioaryloxy and the like. Such heteroaryl groups can have a single ring (e.g., pyridyl or furyl) or multiple condensed rings (e.g., indolizinyl or benzothienyl). Preferred heteroaryls include pyridyl, pyrrolyl and furyl.
xe2x80x9cHeterocyclexe2x80x9d or xe2x80x9cheterocyclicxe2x80x9d refers to a monovalent saturated or unsaturated group having a single ring or multiple condensed rings, from 1 to 8 carbon atoms and from 1 to 4 hetero atoms selected from nitrogen, sulfur or oxygen within the ring.
Unless otherwise constrained by the definition for the heterocyclic substituent, such heterocyclic groups can be optionally substituted with 1 to 3 substituents selected from the group consisting of alkyl, alkoxy, aryl, aryloxy, halo, nitro, heteroaryl, thioalkoxy, thioaryloxy and the like. Such heterocyclic groups can have a single ring (e.g., piperidinyl or tetrahydrofuryl) or multiple condensed rings (e.g., indolinyl, dihydrobenzofuran or quinuclidinyl). Preferred heterocycles include piperidinyl, pyrrolidinyl and tetrahydrofuryl.
Examples of heterocycles and heteroaryls include, but are not limited to, furan, thiophene, thiazole, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, pyrrolidine, indoline and the like.
xe2x80x9cThiolxe2x80x9d refers to the group xe2x80x94SH.
xe2x80x9cThioalkoxyxe2x80x9d refers to the group xe2x80x94S-alkyl.
xe2x80x9cThioaryloxyxe2x80x9d refers to the group aryl-Sxe2x80x94 wherein the aryl group is as defined above including optionally substituted aryl groups as also defined above.
xe2x80x9cThioheteroaryloxyxe2x80x9d refers to the group heteroaryl-Sxe2x80x94 wherein the heteroaryl group is as defined above including optionally substituted aryl groups as also defined above.
The class of biological reagent having the described properties are defined by formula I below:
Axe2x80x94Bxe2x80x94Cxe2x80x83xe2x80x83I
wherein
A is selected from the group consisting of compounds defined by formulas II, III, IV and V as described below;
B is selected from the group consisting of
a) (CH2CH(R)Q)n 
b) alkylene-Q
c) substituted alkylene-Q
wherein R is selected from hydrogen, alkyl, aryl and Q is selected from xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94OC(O)NHxe2x80x94, xe2x80x94NHC(O)Oxe2x80x94, xe2x80x94NHC(O)NHxe2x80x94, xe2x80x94NHC(O)xe2x80x94 and xe2x80x94C(O)NHxe2x80x94 and
C is selected from the group consisting of a solid support and a detectable marker optionally linked to Q through a linking arm.
In one embodiment, A comprises a group defined by Formula II below:: 
wherein R11 is selected from the group consisting of:
(a) a substituted phenyl group of the formula: 
xe2x80x83wherein
Rc is selected from the group consisting of acyl, alkyl, alkoxy, alkoxycarbonyl, alkylalkoxy, azido, cyano, halo, hydrogen, nitro, trihalomethyl, thioalkoxy, and wherein Rb and Rc are fused to form a heteroaryl or heterocyclic ring with the phenyl ring wherein the heteroaryl or heterocyclic ring contains from 3 to 8 atoms of which from 1 to 3 are heteroatoms independently selected from the group consisting of oxygen, nitrogen and sulfur;
Rb and Rbxe2x80x2 are independently selected from the group consisting of hydrogen, halo, nitro, cyano, trihalomethyl, alkoxy, and thioalkoxy with the proviso that Rb, Rbxe2x80x2 and Rc are not all hydrogen and with the further proviso that when Rc is hydrogen, then neither Rb nor Rbxe2x80x2 are hydrogen;
(b) 2-naphthyl; and
(c) 2-naphthyl substituted at the 4, 5, 6, 7 and/or 8 positions with 1 to 5 substituents selected from the group consisting of alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy;
R12 is selected from the group consisting of hydrogen, alkyl of from 1 to 4 carbon atoms, alkylalkoxy of from 1 to 4 carbon atoms and alkylthioalkoxy of from 1 to 4 carbon atoms; and
R13 is selected from the group consisting of xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94Oxe2x80x94(CH2)pC(O)xe2x80x94, xe2x80x94Oxe2x80x94(CH2)pC(O)Oxe2x80x94 and xe2x80x94Oxe2x80x94(CH2)pC(O)NHxe2x80x94, wherein p is an integer of from 1 to 2.
In formula II above, R11 substituted phenyls are preferably 4-substituted, 3,5-disubstituted or 3,4-disubstituted phenyl substituents wherein the substituents at the 3 and/or 5 positions are defined by Rb, Rbxe2x80x2 as above and the substituent at the 4 position is defined by Rc as above. Particularly preferred 3,5-disubstituted phenyls include, by way of example, 3,5-dichlorophenyl, 3,5-difluorophenyl, 3,5-di(trifluoromethyl)phenyl, 3,5-dimethoxyphenyl, and the like. Particularly, preferred 3,4-disubstituted phenyls include, by way of example, 3,4-dichlorophenyl, 3,4-difluorophenyl, 3-(trifluoromethyl)-4-chlorophenyl, 3-chloro-4-cyanophenyl, 3-chloro-4-iodophenyl, 3,4-methylenedioxyphenyl, 3,4-ethylenedioxyphenyl, and the like. Particularly preferred 4-substituted phenyls include, by way of example, 4-azidophenyl, 4-bromophenyl, 4-chlorophenyl, 4-cyanophenyl, 4-ethylphenyl, 4-fluorophenyl, 4-iodophenyl, 4-(phenylcarbonyl)phenyl, 4-(1-ethoxy)ethylphenyl, 4-(ethoxycarbonyl)phenyl, and the like.
In the compounds of formula II, Rb and Rc can be fused to form a heteroaryl or heterocyclic ring with the phenyl ring. Fusion in this manner results in a fused bicyclic ring structure of the formula: 
where Rbxe2x80x2 is as defined above and A is the fused heteroaryl or heterocyclic group containing from 3 to 8 atoms of which from 1 to 3 are heteroatoms independently selected from the group consisting of oxygen, nitrogen and sulfur wherein the two atoms of the phenyl ring are included in the total atoms present in the heteroaryl or heterocyclic group. Examples of such fused ring systems include, for instance, indol-5-yl, indol-6-yl, thionaphthen-5-yl, thionaphthen-6-yl, isothionaphthen-5-yl, isothionaphthen-6-yl, indoxazin-5-yl, indoxazin-6-yl, benzoxazol-5-yl, benzoxazol-6-yl, anthranil-5-yl, anthranil-6-yl, quinolin-6-yl, quinolin-7-yl, isoquinolin-6-yl, isoquinolin-7-yl, cinnolin-6-yl, cinnolin-7-yl, quinazolin-6-yl, quinazolin-7-yl, benzofuran-5-yl, benzofuran-6-yl, isobenzofuran-5-yl, isobenzofuran-6-yl, and the like.
Other preferred R11 substituents include, by way of example, 2-naphthyl, 2-methylquinolin-6-yl, benzothiazol-6-yl, 5-indolyl, and the like.
Preferably R12 is selected from the group consisting of alkyl of from 1 to 4 carbon atoms, alkylalkoxy of from 1 to 4 carbon atoms and alkylthioalkoxy of from 1 to 4 carbon atoms. Particularly preferred R12 substituents include, by way of example, methyl, ethyl, n-propyl, iso-butyl, and the like.
Preferred R13 substituents include methoxy, ethoxy, iso-propoxy, n-propoxy, n-butoxy, iso-butoxy, cyclopentoxy, allyloxy, 4-methylpentoxy, xe2x80x94Oxe2x80x94CH2-(2,2-dimethyl-1,3-dioxolan-4-yl), xe2x80x94Oxe2x80x94CH2-cyclohexyl, xe2x80x94Oxe2x80x94CH2-(3-tetrahydrofuranyl), xe2x80x94Oxe2x80x94CH2xe2x80x94C(O)O-tert-butyl, xe2x80x94Oxe2x80x94CH2xe2x80x94C(CH3)3, xe2x80x94Oxe2x80x94CH2-xcfx86, xe2x80x94OCH2CH(CH2CH3)2, xe2x80x94O(CH2)3CH(CH3)2, xe2x80x94ONxe2x95x90C(NH2)xcfx86, xe2x80x94ONxe2x95x90C(NH2)CH3, xe2x80x94ONxe2x95x90C(NH2)CH2CH3, xe2x80x94ONxe2x95x90C(NH2)CH2CH2CH3, xe2x80x94ONxe2x95x90C(NH2)-cyclopropyl, xe2x80x94ONxe2x95x90C(NH2)xe2x80x94CH2-cyclopropyl, xe2x80x94ONxe2x95x90C(NH2)-cyclopentyl, xe2x80x94ONxe2x95x90C(NH2)CH2CH(CH3)2, and the like.
In another embodiment, A comprises the compounds of Formula III set forth below: 
wherein R21 is selected from the group consisting of
a) alkyl, alkenyl, alkcycloalkyl, phenyl-(Rd)mxe2x80x94, naphthyl-(Rd)mxe2x80x94 wherein Rd is an alkylene group of from 1 to 8 carbon atoms and m is an integer equal to 0 or 1, cycloalkyl, cycloalkenyl, 3-pyridyl, 4-pyridyl and heteroaryl, other than 3- and 4-pyridyl, of 3 to 10 atoms and 1 to 4 heteroatoms selected from oxygen, sulfur and nitrogen wherein the heteroaryl group is optionally substituted with 1 to 3 substituents selected from the group consisting of alkyl, alkoxy, aryl, aryloxy, halo, nitro, thioalkoxy, and thioaryloxy with the proviso that for such heteroaryls when there is at least one nitrogen heteroatom, there is also at least one oxygen and/or sulfur heteroatom;
(b) a substituted phenyl group of the formula: 
xe2x80x83wherein
R is alkylene of from 1 to 8 carbon atoms,
m is an integer equal to 0 or 1,
Re and Rf are independently selected from the group consisting of hydrogen, hydroxy, fluoro and methyl;
Rg and Rh are independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, cyano, cycloalkyl, halo, heteroaryl, heterocyclic, nitro, trihalomethyl, thioalkoxy, thioaryloxy, thioheteroaryloxy, and xe2x80x94C(O)Rj where Rj is selected from the group consisting of alkyl, aryl, alkoxy and aryloxy; and
Rf is selected from the group consisting of hydrogen, alkyl, aryl, cyano, halo, nitro, and where Rg and Ri are fused to form a methylenedioxy ring with the phenyl ring; and
when Rg and/or Rh and/or Ri is fluoro, chloro, bromo and/or nitro, then Re and/or Rf can also be chloro; and
(c) 1- or 2-naphthyl-(Rk)m-substituted at the 5, 6, 7 and/or 8 positions with 1 to 4 substituents selected from the group consisting alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy wherein Rk is an alkylene group of from 1 to 8 carbon atoms and m is an integer equal to 0 or 1;
R22 is selected from the group consisting of hydrogen, alkyl, phenyl, alkylalkoxy, alkylthioalkoxy;
X is oxygen or sulfur;
Xxe2x80x2 is hydrogen, hydroxy or fluoro; and
Xxe2x80x3 is hydrogen, hydroxy or fluoro, or Xxe2x80x2 and Xxe2x80x3 together form an oxo group.
In formula III above, preferred R21 unsubstituted aryl groups include, for example, phenyl, 1-naphthyl, 2-naphthyl, and the like.
Preferred R21 substituted aryl groups include, for example, monosubstituted phenyls having a single substitution at the 2, 3 or 4 positions where each of the particular subsituents is governed by the respective Re/Rf, Rg/Rh and Ri groups; disubstituted phenyls which include those having two substituents at the 2,3-positions, 2,4-positions, 2,5-positions, 2,6-positions, 3,4-positions, 3,5-positions or 3,6-positions where each of these substituents is governed by the respective Re, Rf, Rg, Rh and Ri groups; and trisubstituted phenyls which include those having three substituents at the 2,3,4-positions, 2,3,5-positions, 2,3,6-positions, 3,4,5-positions and 3,4,6-positions again where each of these substituents is governed by the respective Re, Rf, Rg, Rh and Ri groups. Preferably, the substituted phenyl groups do not include more than 3 substituents.
Examples of substituted phenyls include, for instance, 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, 4-nitrophenyl, 4-methylphenyl, 3-methoxy-phenyl, 3-nitrophenyl, 3-fluorophenyl, 3-chlorophenyl, 3-bromophenyl, 3-thiomethoxyphenyl, 3-methylphenyl, 3-trifluoromethylphenyl, 2-hydroxy-phenyl, 2-methylphenyl, 2-fluorophenyl, 3,4-dichlorophenyl, 3,4-methylene-dioxyphenyl, 3,5-difluorophenyl, 3,5-dichlorophenyl, 2,4-dichlorophenyl, and 2,5-difluorophenyl.
Preferred R21 groups represented by phenyl-Rxe2x80x94 include, by way of example, benzyl, 3-phenylethyl, 4-phenyl-n-propyl, and the like.
Preferred R21 alkyl, alkcycloalkyl, cycloalkyl and cycloalkenyl groups include, by way of example, sec-butyl, cyclopropyl, cyclobutyl, cyclohexyl, cyclopentyl, cyclohex-1-enyl, xe2x80x94CH2-cyclopropyl, xe2x80x94CH2-cyclobutyl, xe2x80x94CH2-cyclohexyl, xe2x80x94CH2-cyclopentyl, xe2x80x94CH2CH2-cyclopropyl, xe2x80x94CH2CH2-cyclobutyl, xe2x80x94CH2CH2-cyclohexyl, xe2x80x94CH2CH2-cyclopentyl, and the like.
Preferred R21 heteroaryls and substituted heteroaryls include, by way of example, pyrid-3-yl, pyrid-4-yl, thien-2-yl, thien-3-yl, benzothiazol-4-yl, 2-phenylbenzoxazol-5-yl, furan-2-yl, benzofuran-2-yl, benzothiophen-3-yl, 2-chlorothien-5-yl, 3-methylisoxazol-5-yl, 2-(phenylthio)thien-5-yl, 6-methoxythiophen-2-yl, 3-phenyl-1,2,4-thiooxadiazol-5-yl, 2-phenyloxazol-4-yl, and the like.
Preferably R22 is selected from the group consisting of alkyl of from 1 to 4 carbon atoms, phenyl, alkylalkoxy of from 1 to 4 carbon atoms and alkylthioalkoxy of from 1 to 4 carbon atoms. Particularly preferred R22 substituents include, by way of example, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, xe2x80x94CH2CH2SCH3, cyclohexyl and phenyl.
When X is oxygen, preferred R23 substituents include, for example, methyl, ethyl, iso-propyl, n-propyl, n-butyl, iso-butyl, cyclopentyl, allyl, iso-but-2-enyl, 3-methylpentyl, xe2x80x94CH2-cyclopropyl, xe2x80x94CH2-cyclohexyl, xe2x80x94CH2-(3-tetrahydrofuranyl), xe2x80x94CH2-thien-2-yl, xe2x80x94CH2(1-methyl)cyclopropyl, xe2x80x94CH2-thien-3-yl, xe2x80x94CH2xe2x80x94C(O)O-t-butyl, xe2x80x94CH2xe2x80x94C(CH3)3, xe2x80x94CH2CH(CH2CH3)2, -2-methylcyclopentyl, -cyclohex-2-enyl, xe2x80x94CH[CH(CH3)2]COOCH3, xe2x80x94CH2CH2N(CH3)2, xe2x80x94CH2C(CH3)xe2x95x90CH2, xe2x80x94CH2CHxe2x95x90C(CH3)2 and the like.
When X is sulfur, preferred R23 substituents include, for example, iso-but-2-enyl and iso-butyl.
In another embodiment, A comprises the compounds set forth in Formula IV below: 
wherein R31 is selected from the group consisting of
(a) phenyl,
(b) a substituted phenyl group of the formula: 
xe2x80x83wherein
Rp is selected from the group consisting of acyl, alkyl, alkoxy, alkylalkoxy, azido, cyano, halo, hydrogen, nitro, trihalomethyl, thioalkoxy, and wherein Rp and Rm are fused to form a heteroaryl or heterocyclic ring with the phenyl ring,
Rm and Rn are independently selected from the group consisting of hydrogen, halo, nitro, cyano, trihalomethyl, alkoxy, and thioalkoxy with the proviso that when RP is hydrogen, then Rm and Rn are either both hydrogen or both substituents other than hydrogen,
(c) 2-naphthyl,
(d) 2-naphthyl substituted at the 4, 5, 6, 7 and/or 8 positions with 1 to 5 substituents selected from the group consisting of alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, thioalkoxy, aryl, and heteroaryl,
(e) heteroaryl, and
(f) substituted heteroaryl containing 1 to 3 substituents selected from the group consisting of alkyl, alkoxy, aryl, aryloxy, cyano, halo, nitro, heteroaryl, thioalkoxy and thioaryloxy provided that said substituents are not ortho (adjacent) to the heteroaryl attachment to the xe2x80x94NH group;
R32 is selected from the group consisting of hydrogen, alkyl of from 1 to 4 carbon atoms, alkylalkoxy of from 1 to 4 carbon atoms, alkylthioalkoxy of from 1 to 4 carbon atoms, aryl, heteroaryl, substituted aryl and substituted heteroaryl provided that the substituents are not ortho (adjacent) to the attachment of the aryl or heteroaryl atom to the carbon atom;
R33 is selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, substituted alkyl, substituted alkenyl, substituted alkynyl, and heterocyclic; and
X is selected from the group consisting of oxygen and sulfur.
In formula IV above, R31 substituted phenyls are preferably 4-substituted, 3,5-disubstituted or 3,4-disubstituted phenyl substituents wherein the substituents at the 3 and/or 5 positions are defined by Rm, Rmxe2x80x2 as above and the substituents at the 4 position is defined by Rl as above. Particularly preferred 3,5-disubstituted phenyls include, by way of example, 3,5-dichlorophenyl, 3,5-difluorophenyl, 3,5-di(trifluoromethyl)phenyl, 3,5-dimethoxyphenyl, and the like. Particularly, preferred 3,4-disubstituted phenyls include, by way of example, 3,4-dichlorophenyl, 3,4-difluorophenyl, 3-(trifluoromethyl)-4-chlorophenyl, 3-chloro-4-cyanophenyl, 3-chloro-4-iodophenyl, 3,4-methylenedioxyphenyl, and the like. Particularly preferred 4-substituted phenyls include, by way of example, 4-azidophenyl, 4-bromophenyl, 4-chlorophenyl, 4-cyanophenyl, 4-ethylphenyl, 4-fluorophenyl, 4-iodophenyl, 4-(phenylcarbonyl)phenyl, 4-(1-ethoxy)ethylphenyl, and the like.
Other preferred R31 substituents include, by way of example, 2-naphthyl, quinolin-3-yl, 2-methylquinolin-6-yl, benzothiazol-6-yl, benzothiazol-2-yl, 5-indolyl, phenyl, 2-naphthyl, and the like.
In the compounds of formula IV, Rp and Rm can be fused to form a heteroaryl or hetero cyclic ring with the phenyl ring. Fusion in this manner results in a fused bicyclic ring structure of the formula: 
where Rn is as defined above and A is the fused heteroaryl or heterocyclic group as these terms are as defined above wherein the two atoms of the phenyl ring are included in the total atoms present in the heteroaryl or heterocyclic group. Examples of such fused ring systems include, for instance, indol-5-yl, indol-6-yl, thionaphthen-5-yl, thionaphthen-6-yl, isothionaphthen-5-yl, isothionaphthen-6-yl, indoxazin-5-yl, indoxazin-6-yl, benzoxazol-5-yl, benzoxazol-6-yl, anthranil-5-yl, anthranil-6-yl, quinolin-6-yl, quinolin-7-yl, isoquinolin-6-yl, isoquinolin-7-yl, cinnolin-6-yl, cinnolin-7-yl, quinazolin-6-yl, quinazolin-7-yl, benzofuran-5-yl, benzofuran-6yl, isobenzofuran-5-yl, isobenzofuran-6-yl, and the like.
Preferably R32 is selected from the group consisting of alkyl of from 1 to 4 carbon atoms, alkylalkoxy of from 1 to 4 carbon atoms, alkylthioalkoxy of from 1 to 4 carbon atoms, aryl, heteroaryl, substituted aryl and substituted heteroaryl provided that the substituents are not ortho to the attachment of the aryl or heteroaryl atom to the carbon atom. Particularly preferred R32 substituents include, by way of example, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, xe2x80x94CH2CH2SCH3, phenyl and the like.
Preferred R33 substituents include alkyl groups such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and the like; substituted alkyl groups such as a-hydroxyethyl, xe2x80x94CH2-cyclohexyl, benzyl, p-hydroxybenzyl, 3-iodo-4-hydroxybenzyl, 3,5-diiodo-4-hydroxybenzyl, xe2x80x94CH2-indol-3-yl, phenyl, xe2x80x94(CH2)4xe2x80x94NHxe2x80x94BOC, xe2x80x94(CH2)4xe2x80x94NH2, xe2x80x94CH2-(1-N-benzyl-imidazol-4-yl), xe2x80x94CH2-imidazol-4-yl, xe2x80x94CH2CH2SCH3, xe2x80x94(CH2)4NHC(O)(CH2)4CH3, xe2x80x94(CH2)yC(O)OR35 where y is 1 or 2 and R35 is hydrogen, methyl, tert-butyl, phenyl, and the like.
In another embodiment, A comprises the compounds represented by Formula V set forth below: 
wherein
R41 is selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkenyl, substituted alkynyl, aryl, heteroaryl and heterocyclic;
R42 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclic;
each R43 is independently selected from the group consisting of hydrogen and methyl and R43 together with R44 can be fused to form a cyclic structure of from 3 to 8 atoms which is optionally fused with an aryl or heteroaryl group;
each R44 is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, heterocyclic, substituted alkyl, substituted alkenyl and substituted alkynyl;
each R45 is selected from hydrogen and methyl or together with R43 forms a cycloalkyl group of from 3 to 6 carbon atoms;
X is selected from oxygen, sulfur and NH;
Xxe2x80x2 is hydrogen, hydroxy or fluoro; and
Xxe2x80x3 is hydrogen, hydroxy or fluoro, or Xxe2x80x2 and Xxe2x80x3 together form an oxo group;
Z is selected from the group consisting of a bond covalently linking R41 to xe2x80x94CXxe2x80x2Xxe2x80x3xe2x80x94, oxygen and sulfur; and
n is an integer equal to 1 to 3.
In formula V above, Xxe2x80x3 is preferably hydrogen and Xxe2x80x2 is preferably hydrogen or fluoro.
In formula V above, Z is preferably a covalent bond linking Rl to xe2x80x94CXxe2x80x2Xxe2x80x3xe2x80x94.
In formula V above, preferred R41 unsubstituted aryl groups include, for example, phenyl, 1-naphthyl, 2-naphthyl, and the like.
Preferred R41 substituted aryl groups include, for example, monosubstituted phenyls (preferably 3 or 5 substituents); disubstituted phenyls (preferably 3,5 substituents); and trisubstituted phenyls (preferably 3,4,5 substituents). Preferably, the substituted phenyl groups do not include more than 3 substituents.
Examples of substituted phenyls include, for instance, 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, 4-nitrophenyl, 4-methylphenyl, 3-methoxy-phenyl, 3-nitrophenyl, 3-fluorophenyl, 3-chlorophenyl, 3-bromophenyl, 3-thiomethoxyphenyl, 3-methylphenyl, 3-trifluoromethylphenyl, 2-hydroxy-phenyl, 2-methylphenyl, 2-fluorophenyl, 2-chlorophenyl, 3,4-difluorophenyl, 2,3,4,5,6-pentafluorophenyl, 3,4-dibromophenyl, 3,4-dichlorophenyl, 3,4-methylene-dioxyphenyl, 3,5-difluorophenyl, 3,5-dichlorophenyl, 2,4-dichlorophenyl, and 2,5-difluorophenyl.
Preferred R41 alkaryl groups include, by way of example, benzyl, 2-phenylethyl, 3-phenyl-n-propyl, and the like.
Preferred R41 alkyl, substituted alkyl, alkenyl, cycloalkyl and cycloalkenyl groups include, by way of example, iso-propyl, n-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, xe2x80x94CH2CHxe2x95x90CH2, xe2x80x94CH2CHxe2x95x90CH(CH2)4CH3, cyclopropyl, cyclobutyl, cyclohexyl, cyclopentyl, cyclohex-1-enyl, xe2x80x94CH2-cyclopropyl, xe2x80x94CH2-cyclobutyl, xe2x80x94CH2-cyclohexyl, xe2x80x94CH2-cyclopentyl, xe2x80x94CH2CH2-cyclopropyl, xe2x80x94CH2CH2-cyclobutyl, xe2x80x94CH2CH2-cyclohexyl, xe2x80x94CH2CH2-cyclopentyl, aminomethyl, N-tert-butoxycarbonylaminomethyl, and the like.
Preferred R41 heteroaryls and substituted heteroaryls include, by way of example, pyrid-2-yl, pyrid-3-yl, pyrid-4-yl, fluoropyridyls (including 5-fluoropyrid-3-yl), chloropyridyls (including 5-chloropyrid-3-yl), thien-2-yl, thien-3-yl, benzothiazol-4-yl, 2-phenylbenzoxazol-5-yl, furan-2-yl, benzofuran-2-yl, thionaphthen-2-yl, 2-chlorothiophen-5-yl, 3-methylisoxazol-5-yl, 2-(thiophenyl)thiophen-5-yl, 6-methoxythionaphthen-2-yl, 3-phenyl-1,2,4-thiooxadiazol-5-yl, 2-phenyloxazol-4-yl, and the like.
Preferably R42 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, aryl, heteroaryl and heterocyclic. Particularly preferred R42 substituents include, by way of example, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, phenyl, 4-fluorophenyl, 3,5-difluoro-phenyl, 4-methoxyphenyl, benzyl, cyclopropyl, cyclohexyl, cyclopentyl, cycloheptyl, thien-2-yl, thien-3-yl, xe2x80x94CH2CH2SCH3, xe2x80x94CH2OCH2xcfx86, xe2x80x94CH(CH3)OCH2xcfx86, xe2x80x94CH(OH)CH3, xe2x80x94CH2OH and the like. As noted below, R42 (as well as R44) is preferably the side chain of an L-amino acid.
Preferably, R43 is hydrogen, methyl or together with R44 and the nitrogen to which R43 is attached forms pyrrolidin-2-yl, 2,3-dihydroindol-2-yl, piperidin-2-yl, 4-hydroxy-pyrrolidin-2-yl, 1,2,3,4-tetrahydroisoquinolin-3-yl, and the like.
Preferred R44 substituents include, for example, hydrogen, methyl, ethyl, iso-propyl, n-propyl, n-butyl, sec-butyl, iso-butyl, cyclopentyl, cyclohexyl, allyl, iso-but-2-enyl, 3-methylpentyl, xe2x80x94CH2-cyclopropyl, xe2x80x94CH2-cyclohexyl, xe2x80x94CH2-indol-3-yl, phenyl, p-(phenyl)phenyl, m-(phenyl)phenyl o-fluorophenyl, m-fluorophenyl, p-fluorophenyl, p-bromophenyl, m-methoxyphenyl, p-methoxyphenyl, phenethyl, benzyl, m-hydroxybenzyl, p-hydroxybenzyl, p-nitrobenzyl, m-trifluoromethylphenyl, p-(CH3)2NCH2CH2CH2O-benzyl, p-(CH3)3COC(O)CH2O-benzyl, p-phenylphenyl, 3,5-difluorophenyl, p-(HOOCCH2O)-benzyl, 2-aminopyrid-6-yl, 4xe2x80x94(N-morpholino-CH2CH2O)-benzyl, xe2x80x94CH2CH2C(O)NH2, xe2x80x94CH2-imidazol-4-yl, xe2x80x94CH2-(3-tetrahydrofuranyl), xe2x80x94CH2-thien-2-yl, xe2x80x94CH2-thiazol-4-yl, xe2x80x94CH2(1-methyl)cyclopropyl, xe2x80x94CH2-thien-3-yl, thien-3-yl, thien-2-yl, xe2x80x94CH2xe2x80x94C(O)Oxe2x80x94t-butyl, xe2x80x94CH2xe2x80x94C(CH3)3, xe2x80x94CH2CH(CH2CH3)2, 2-methylcyclopentyl, -cyclohex-2-enyl, xe2x80x94CH[CH(CH3)2]COOCH3, xe2x80x94(CH2)2SCH3, xe2x80x94CH2CH2N(CH3)2, xe2x80x94CH2C(CH3)xe2x95x90CH2, xe2x80x94CH2CHxe2x95x90CHCH3 (cis and trans), xe2x80x94CH2OH, xe2x80x94CH(OH)CH3, xe2x80x94CH(Oxe2x80x94t-butyl)CH3, xe2x80x94CH2OCH3, xe2x80x94(CH2)4NH-Boc, xe2x80x94(CH2)4NH2, xe2x80x94(CH2)4N(CH3)2, xe2x80x94CH2-pyridyl (e.g., 2-pyridyl, 3-pyridyl and 4-pyridyl), pyridyl (2-pyridyl, 3-pyridyl and 4-pyridyl), xe2x80x94CH2-naphthyl (e.g., 1-naphthyl and 2-naphthyl), xe2x80x94CH2xe2x80x94(N-morpholino), pxe2x80x94(N-morpholino-CH2CH2O)xe2x80x94benzyl, benzo[b]thiophen-2-yl, benzo[b]thiophen-3-yl, 5-chlorobenzo[b]thiophen-2-yl, 4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl, benzo[b]thiophen-3-yl, tetrazol-5-yl, 5-chlorobenzo[b]thiophen-3-yl, benzo[b]thiophen-5-yl, 6-methoxynaphth-2-yl, xe2x80x94CH2xe2x80x94N-phthalimidyl, 2-methylthiazol4-yl, and thieno[2,3-b]thiophen-2-yl, 5-bromothien-2-yl, 4-bromothien-2-yl, 5-chlorothien-2-yl, 3-phenoxyphenyl, 2-phenoxyphenyl, 4-ethylphenyl, 2-benzylphenyl, (4-ethylphenyl)phenyl, 4-tert-butylphenyl, 4-n-butylphenyl, o-(4-chlorophenoxy)phenyl, furan-2-yl, 4-phenylacetylenylphenyl and the like.
Preferably, R45 is hydrogen. However, in another embodiment, R44 and R45 are fused to form a cycloalkyl group including, for example, cyclopropyl, cyclobutyl, and the like.
xe2x80x9cDetectable markerxe2x80x9d means a radioactive label, a fluorescent label, a chemiluminescer, a heavy metal ion, an antibody, an enzyme, biotin, an azido group, an immunobiotin and the like.
Conveniently, a radioactive label may be employed. Radioactive labels include 125I, 32P, 3H, 14C and the like. Any radioactive label may be employed which provides for an adequate signal and has sufficient half-life.
Further, a fluorescent label may be employed. Fluorescent labels include fluorescein, rhodamine and its derivatives, dansyl, umbelliferone, BODIPY(copyright), and the like. Any fluorescent label may be employed with provides for an adequate signal. A chemiluminescer may be employed. Suitable chemiluminescers include luciferin, and 2,3-dihydrophthalazinediones, i.e. luminol.
Other labels include ligands, which can serve as a specific binding pair member to a labeled antibody, fluorescers, chemiluminescers, enzymes, photoaffinity probes, antibodies which can serve as a specific binding pair member for a labeled ligand and the like. A wide variety of labels have been employed in immunoassays which can be readily employed in the present methods. The choice of label will be governed by the effect of the label on the rate of binding of the biological reagent to the target. It will be necessary that the label provide sufficient sensitivity to detect the target.
xe2x80x9cSolid supportxe2x80x9d means any solid support in which a compound can be affixed. Examples of these supports include glass, test tubes, microtiter plates, nylon beads, agarose beads, magnetic beads, glass beads, teflon, polystyrene beads, photodetectable chips and the like.
Preferably, the solid support or the detectable marker contain a reactive functional group which is complementary to and reacts with the compounds of Formula VI to form the compounds of Formula I.
xe2x80x9cLinking armsxe2x80x9d are well known in the art and include, by way of example only, conventional linking arms such as those comprising ester, amide, carbamate, ether, thio ether, urea, amine groups and the like. The linking arm can be cleavable or non-cleavable.
Cleavable linking arms refer to linking arms wherein at least one of the covalent bonds of the linking arm which attaches the compound t o the solid support can be readily broken by specific chemical reactions thereby providing for compounds free of the solid support or detectable marker. The chemical reactions employed to break the covalent bond of the linking arm are selected so as to be specific for bond breakage thereby preventing unintended reactions occurring elsewhere on the compound. The cleavable linking arm is selected relative to the synthesis of the compounds to b e formed on the solid support so as to prevent premature cleavage of this compound from the solid support as well as not to interfere with any of the procedures employed during compound synthesis on the support. Suitable cleavable linking arms are well known in the art.
xe2x80x9cNon-cleavable linking armsxe2x80x9d refer to linking arms wherein the covalent bond(s) linking the activated ketone compound to the solid support can only be cleaved under conditions which chemically alters unintended parts of the structure of the compound attached thereto.
Compound Preparation
The compounds of formula I above are readily prepared via several divergent synthetic routes with the particular route selected relative to the ease of compound preparation, the commercial availability of starting materials, and the like.
The starting materials for compounds comprising Formula II are readily prepared by the methods set forth in International Patent Application No. PCT/US97/203568, which is incorporated by reference herein in its entirety.
The starting materials for compounds comprising Formula III are readily prepared by the methods set forth in International Patent Application No. PCT/US97/203559, which is incorporated by reference herein in its entirety.
The starting materials for compounds comprising Formula IV are readily prepared by the methods set forth in International Patent Application No. PCT/US97/1870410, which is incorporated by reference herein in its entirety.
The starting materials for compounds comprising Formula V are readily prepared by the methods set forth in International Patent Application No. PCT/US97/2080411, which is incorporated by reference herein in its entirety.
The manner in which the label is bound to the compound will vary depending upon the nature of the label. For a radioactive label, a wide variety of techniques can be employed which are known in the art.
The manner for attaching the biological reagent to a solid support is well known in the art. One method is by derivatizing the end of the linker having a carboxyl or amino group. Another example of attachment is by attaching a biotin molecule to the biological reagent and then attaching the biological reagent to a solid support bearing avidin molecules.
It is recognized that the biological reagent should be attached to the solid support in an orientation which will allow binding of the biological reagent to the protein or peptide of interest.
Methods
The biological reagents of the present invention are useful in determining the mechanism of xcex2-amyloid peptide release and/or its synthesis, and, accordingly, have utility in future drug discovery.
Without being limited to a theory, it is thought that the xcex2-amyloid peptide is generated by the actions of two enzymes, a xcex2-secretase which cleaves the parent protein at the amino terminus of the xcex2-amyloid peptide and a xcex3-secretase which cleaves the parent protein at the carboxyl terminus of the xcex2-amyloid peptide. It is thought that the compounds of formula I, II, III and IV act to inhibit the cleavage by the xcex3-secretase, either directly or indirectly via a protein modulating xcex3-secretase activity.
The biological reagents are useful in identifying the cellular factor (either peptide, protein etc.) whose activity is inhibited by the compounds of formula I, II, III and IV. Such biological reagents can be used is a variety of ways depending on the reactive group which comprises C in Formula I.
In one embodiment, cells known to produce xcex3-amyloid peptide are disrupted to produce a cell membrane suspension. Such cellular membrane suspensions are passed over the biological reagents of the present invention, wherein C of formula I comprises a solid support. The cellular factor or factors which interacts with the compounds of formula II, III, IV and V, will bind to the biological reagents of the present invention. The remainder of the cellular biological solution will be washed from the biological reagent comprising the solid support. The cellular factor can then be identified.
In another embodiment, cells known to produce xcex2-amyloid peptide are disrupted to produce a cellular membrane suspension. Such cellular membrane suspensions are mixed with the biological reagents of the present invention, wherein C of formula I comprises a photoaffinity reagent, for example an azido group under conditions whereby the cellular factor or factor which interact with the compounds of formula II, III, IV and V will bind. The mixture is then subjected to light, for example, ultraviolet light which covalently links the biiological reagents of the present invention to the cellular factor. The cellular factor bound to the photoaffinity reagent is then removed/purified from the remainder of the biological solution and the cellular factor identified.
In another embodiment, cells known to produce xcex2-amyloid peptide are mixed with the biological reagent of the present invention wherein C of Formula I comprises a fluorescent dye under conditions wherein the biological reagent of the present invention binds to cells comprising the cellular factor or factors involved in the production of xcex2-amyloid peptide. Such cells are then identified by their fluorescence. Methods of identification include, but are not limited to, cell sorting.
It is contemplated that the cells known to produce xcex2-amyloid peptide may include a library of eukaryotic or prokaryotic cells transformed with genes under the control of an expression vector, such that the genes are expressed in the cells. Cells expressing the cellular factor or factors involved in the production of the xcex2-amyloid peptide may be identified by fluorescence. The isolated cells may then be disrupted and the expressed gene and or cellular factor identified.
The cellular factor may be identified by an number of methods, including, without being limited to, peptide sequencing, binding to known antibodies and the like. The gene coding for the cellular factor may be sequenced. By these methods the cellular factor involved in xcex2-amyloid peptide release and/or its synthesis may be identified.
Utility
The compounds of the invention are useful in determining the mechanism for xcex2-amyloid peptide release and/or its synthesis, and, accordingly, have utility in future drug discovery.
In particular the biological reagents may be used to determine cellular agents, such as peptides or proteins involved with xcex2-amyloid peptide release and/or its synthesis. Such identification involves the binding of the biological reagent to the cellular agent in a solution and the extraction of the biological reagent/cellular agent from the solution and the identification of the cellular agent.
The biological reagents may also be used to identify cells which express the cellular agent, such as peptides or protein involved with xcex2amyloid peptide release and/or its synthesis. Such identification would involve labeling of cells expressing the cellular agent by attachment of detectably labelled biological reagents of the present invention to such cells.
The following synthetic and biological examples are offered to illustrate this invention and are not to be construed in any way as limiting the scope of this invention. Unless otherwise stated, all temperatures are in degrees Celsius.